Abiotic stresses, such as drought, extreme temperature, high salinity and nutrient starvation, are major environmental factors that limit plant growth and productivity worldwide. For example, in the United States, approximately 90% of lands are constantly subjected to abiotic stresses, and approximately 70% of the maximal potential yields of major crops are routinely lost due to the unfavorable environmental factors (Boyer (1982), Science 218: 443-448). To circumvent environmental stresses, many plants have developed different physiological and biochemical mechanisms to adapt or tolerate stress conditions. Accumulation of low-molecular-weight osmolytes, such sugar alcohols, special amino acids and Gly betaine (Greenway and Munns (1980), Annu Rev Plant Physiol 31: 149-190; Yancey et al. (1982), Science 217: 1214-1222) or expression of some new proteins, e.g., late embryogenesis abundant (LEA) proteins (Greenway and Munns (1980), above; Yancey et al. (1982), above; Baker et al. (1988), Plant Mol Biol 11: 277-291; Dure et al. (1989), Plant Mol Biol 12: 475-486; Skriver and Mundy (1990), Plant Cell 2: 503-512; Chandler and Robertson, (1994) Annu Rev Plant Physiol Plant Mol Biol 45:113-141), have been suggested to play roles in stress tolerance of plants.
Compared with traditional plant breeding, genetic engineering provides a relatively fast and precise means of achieving improved stress tolerance of crop plants. Over-accumulation of osmolytes, stress-regulated transcription factors, transporters that maintain ionic homeostasis, oxidative stress-related proteins, or LEA proteins in transgenic plants have been widely pursued as effective strategies in improving plant tolerance to a broad range of stresses (Bajaj et al. (1999), Mol Breeding 5: 493-503; Bartels (2001), Trends Plant Sci 6: 284-286; Ho and Wu (2004), In H. T. Nguyen and A. Blum, ed., Physiology and Biotechology Integration for Plant Breeding. Marcel Dekker, Inc, New York, N.Y., pp. 489-502). However, under normal environmental conditions, constitutive over-production of these compounds or proteins consumes extra energy and often results in suboptimal growth, altered metabolisms and/or productivity of plants (Goddijn et al. (1997), Plant Physiol 113: 181-190; Romero et al. (1997), Planta 201:293-297; Kasuga et al. (1999), Nat Biotechnol 17: 287-291; Hsieh et al. (2002), Plant Physiol 130: 618-626). Thus, it is desirable to generate transgenic plants that synthesize a high level of an osmoprotectant or a protein, or initiate any other stress tolerant mechanisms, only under a stressful condition.
To minimize the negative effects of transgene overepression on growth and productivity while improving stress-tolerance of plants, the use of stress-responsive promoters has been demonstrated to be a promising approach (Su et al. (1998) Plant Physiol 117:913-922; Kasuga et al. (1999), Nat Biotechnol 17: 287-291; Garg et al. (2002), Proc Natl Acad Sci USA 99: 15898-15903; Lee et al. (2003), Plant Cell Environ 26: 1181-1190; Fu et al. (2007), Plant Cell Rep (in press)).
Abscisic acid (ABA) regulates the expression of many genes that may function in the adaptation of vegetative tissues to several abiotic stresses as well as in seed maturation and dormancy (Himmelbach et al. (2003), Curr Opin Plant Biol 6: 470-479; Shinozaki et al. (2003), Curr Opin Plant Biol 6: 410-417; Taiz and Zeiger (2006) Chapter 23, In: Plant Physiology, 4th edition. Sinauer Associates, Inc., pp. 594-613; Yamaguchi-Shinozaki and Shinozaki (2006). Annu Rev Plant Biol 57: 781-803). Many ABA-inducible genes contain a conserved ABA responsive cis-acting element with an ACGT core, designated as ABRE or G box, in their promoters (Guiltinan et al. (1990), Science 250:267-271; Skriver et al. (1991), Proc Natl Acad Sci USA 88: 7266-7270; Shen et al. (1993), J Biol Chem 268:23652-23660). Promoter studies of two barley ABA inducible genes, HVA1 and HVA22, indicated that ABRE and another cis-acting coupling element (CE), together forming an ABA response complex (ABRC), are required for high-level ABA-induced gene transcription (Straub et al. (1994), Plant Mol Biol 26: 617-630; Shen and Ho (1995), Plant Cell 7: 295-307; Shen et al. (1996), Plant Cell 8: 1107-1119).
The ABRC from HVA22 (ABRC1) is composed of ABRE3 or A3 and a downstream coupling element CE1 (A3-CE1). The ABRC from HVA1 (ABRC3) is composed of ABRE2 or A2 and an upstream coupling element CE3 (CE3-A2) (Shen et al. (1996), above). Studies with a barley aleurone transient expression assay system indicated that the ABRE3 (A3) from HVA22 is interchangeable with the ABRE2 from HVA1 for conferring ABA inducible response, suggesting that both ABREs could interact with either CE1 from HVA22 or CE3 from HVA1, while CE1 from HVA22 is not fully exchangeable with CE3 from HVA1 (Shen et al. (1996), above). Nevertheless, the presence of both CE1 and CE3 accompanying ABRE2 (A2) or ABRE3 has a synergistic effect on the absolute activity as well as on the ABA induction of a promoter (Shen et al. (1996), above). Furthermore, in both leaves and aleruone tissues, the HVA1 ABRC3 has a higher absolute activity and is more responsive to ABA as compared to the HVA22 ABRC1 (Shen et al. (1996), above).
Both ABRC1 and ABRC3 have been used to control stress-inducible expression of foreign genes in both monocot and dicot transgenic plants. For example, fusion of one or four copies of ABRC 1 to the rice Act1 minimal promoter confers induced expression of a reporter gene in a transgenic rice plant, a monocot plant, by ABA, dehydration or salt (Su et al. (1998), above). Expression of two E. coli trehalose biosynthetic genes in a transgenic rice plant, under the control of a promoter containing four copies of ABRC1, led to accumulation of trehalose and improved growth of these plants under salt, drought and low-temperature stress conditions (Garg et al. (2002), above). Expression of an Arabidopsis transcription factor CBF1 in a transgenic tomato plant, a dicot plant, under the control of a promoter containing three copies of ABRC1 fused to the barley α-amylase gene (Amy64) minimal promoter, has also been shown to improve plant growth under chilling, dehydration and salt conditions, while maintain normal growth and productivity under normal growth conditions (Lee et al. (2003), above). Expression of HVA1 in transgenic creeping bentgrass, under the control of a promoter containing two copies of ABRC3, also led to the accumulation of HVA1 and lessened water-deficit injury in these plants (Fu et al. (2007), above).
LEA proteins are a set of proteins highly accumulated in embryos at the late stage of seed development (Dure (1981), Biochemistry 20: 4162-4168; Dure (1992), In DPS Verma, ed., Control of Plant Gene Expression, CRC Press, Boca Raton, Fla., pp. 325-335). LEA proteins were initially classified into three major groups based on conservation in amino acid sequence domains (Baker et al. (1988), Plant Mol Biol 11: 277-291; Dure et al. (1989), Plant Mol Biol 12: 475-486). The conserved domains in group 3 LEA proteins are composed of tandem repeats of an 1′-amino acid motif that may form an amphiphilic α-helix structure (Baker et al. (1988), above; Dure et al. (1989), above; Dure (1993), Plant J 3: 363-369). The correlation between the LEA protein accumulation and stress tolerance has been demonstrated in a number of plants. For example, levels of group 3 LEA proteins were elevated in seedlings of dehydration tolerant wheat (Ried and Walker-Simmons (1993), Plant Physiol 102: 125-131) and roots of ABA and salt tolerant rice (Moons et al. (1995), Plant Physiol 107:177-186).
HVA1 is a member of the group 3 LEA proteins identified in barley aleurone, which is specifically expressed in aleurone layers and embryos during late seed development undergoing desiccation (Hong et al. (1988), Plant Mol Biol 11: 495-506). The expression of HVA1 is rapidly induced in young seedlings by ABA and several stressful conditions, including dehydration, salt, and extreme temperatures (Hong et al. (1992), Plant Mol Biol 18: 663-674; Sutton et al. (1992), Plant Physiol 99: 338-340; Straub et al. (1994), Plant Mol Biol 26: 617-630). Function of HVA1 in protection against drought, salt and/or osmotic stresses has been demonstrated by transgenic approaches in rice (Xu et al. (1996), Plant Physiol 110: 249-257) wheat (Sivamani et al. (2000), Plant Science 155: 1-9) oat (Maqbool et al. (2002), Theor Appl Genet. 105: 201-208) and bentgrass (Fu et al. (2007), above).
As the world's population increases at a high rate, the improvement of crop productivity remains an important and challenging task. Development of crops that are more tolerant to various abiotic stresses could lead to the use of more new lands for cultivation. There remains a need for development of crops that are more tolerant to various abiotic stresses and new genetic tools that enable such development. The present invention satisfies this need.